Leishmania, a protozoan pathogen, is the causative agent of various forms of leishmaniasis like cutaneous, mucocutaneous and visceral leishmaniasis; of which visceral Leishmaniasis is fatal (Kedzierski, et al., 2009). Leishmaniasis affects about 12 million people worldwide and 500,000 new cases are reported annually (WHO bulletin 2012). Drugs used for chemotherapy of leishmaniasis, such as antimonials, miltefosine, paromomycin and amphotericin B are very toxic, expensive and frequent resistance occurs against these drugs in endemic areas (Croft & Coombs, 2003). Moreover, no effective vaccine against leishmaniasis is available (WHO bulletin 2012). Now, it is well established that this disease is associated with the lack of Th1 response characterized by down-regulation of IL-12, IFN-γ while disease promoting Th2 cytokines like IL-4, IL-10 are up-regulated (Bacellar, et. al., 2000; Nylen & Sacks, 2007). Therefore, the major focus is to generate protective T-cell responses using appropriate antigen.
Thus, there is an immense requirement to develop an effective vaccine against leishmaniasis. Recently, considerable progress has been made and large numbers of Leishmania antigens have been tried as potential vaccine candidates including surface expressed antigens like gp63, gp46, PSA-2, receptors of activated C kinase (LACK), cysteine proteases (CP), kinetoplastid membrane protein-11 (KMP-11) etc with varied immune response and diverse species specific protection (Kedzierski, 2010; Singh & Sundar, 2012). However, vaccines protective against leishmaniasis are still not available. Therefore, there is need to identify a novel antigen which is physiologically important for the biology of the parasite.
In addition, no cost effective appropriate diagnostic procedure for detection of leishmaniasis is available. Microscopic examination of the Giemsa stained lesion biopsy smears, lymph node, bone marrow or spleen aspirates remain the standard test in areas of endemicity which require hospitalization. Moreover, owing to the low socio-economic status of the people who are largely affected by these diseases, the tests need to be simple and affordable. Molecular approaches like PCR for parasite detection, though specific, remain restricted to hospitals and research centers owing to its high cost (Franceschi et al., 2007). Recently, a specific immunochromatographic test has been developed based on k39 (a repetitive immunodominant epitope in a kinesin-related protein that is highly conserved among viserotropic Leishmania species) and sufficiently validated for field use (Hailu, 2002; Mathur et al., 2005). The test checks for the presence of anti-k39 IgG/M antibodies in serum and has shown excellent sensitivity (93-100%) and specificity (97-98%) in many VL-endemic countries (Alborzi et al., 2006; Pedras et al., 2008). However, there is a need to develop simple and cost effective diagnostic procedure against Leishmania infection.
Leishmania require heme from exogenous sources for growth due to lack of complete heme biosynthetic pathway (Sah, et. al., 2002). As heme, a critical prosthetic group required by the parasites for several metabolic pathways, thus, heme acquisition process in Leishmania could be a potential target (Kelly, et. al., 2003). Leishmania endocytosed hemoglobin (Hb) through a high affinity hemoglobin receptor (HbR) located on the cell surface (Sengupta, et. al., 1999) and internalized Hb is targeted to the lysosomal compartment where it is degraded to generate intracellular heme (Singh, et. al., 2003; Patel, et. al., 2008) which is used by Leishmania for their survival. This receptor is a surface localized hexokinase and N-terminal end of the receptor is extracellular hemoglobin binding region (Krishnamurthy, et. al., 2005). Thus, this molecule has dual roles; firstly, it acts as Hb receptor on cell surface and secondly, being hexokinase, it regulates glycolysis in parasites. Thus, HbR is bi-functional molecule performing two major functions in Leishmania. The present invention relates to active or inactive hemoglobin receptor or its parts either in DNA or protein forms as a novel immunogen for vaccine against leishmaniasis in mammals.
The present innovation demonstrates that HbR-DNA offers following major advantages over other vaccine candidates against VL.                a. HbR-DNA vaccination induces sterile protection against Leishmania infection in both mice and hamster.        b. It not only evokes protective Th1-response without any adjuvant but also suppresses disease promoting cytokines to confer sterile protection.        c. HbR is a bifunctional antigen possibly interferes with two major pathways in Leishmania.         d. Anti-HbR antibody inhibits both promastigote and amastigote growth, therefore, anti-HbR antibody present in vaccinated animals might kills extracellular parasites during infection process.        e. Successful protection of VL with vaccination of truncated HbR (HbR-N) indicates the feasibility of developing subunit vaccine of HbR against leishmaniasis.        f. HbR is conserved across Leishmania species and hence could also be potential candidate against different forms of leishmaniasis.        g. HbR protein or fragments thereof as marker for diagnosis of Leishmania in kala-azar patients.        